Hydroxy-tryptophan containing derivatives of tritrpticin: modification of antimicrobial activity and membrane interactions.

Tritrpticin is an antimicrobial peptide with a strong microbicidal activity against Gram-positive and Gram-negative bacteria as well as fungi. The 13-residue peptide is essentially symmetrical and possesses a unique cluster of three Trp residues near the center of its amino acid sequence. The mechanism of action of tritrpticin is believed to involve permeabilization of the cytoplasmic membrane of susceptible bacteria. However it has been suggested that intracellular targets may also play a role in its antimicrobial activity. In this work the mechanism of action of several tritrpticin derivatives was studied through substitution of the three Trp residues with 5-hydroxy-tryptophan (5OHW), a naturally occurring non-ribosomal amino acid. Although it is more polar, 5OHW preserves many of the biophysical and biochemical properties of Trp, allowing the use of fluorescence spectroscopy and NMR techniques to study the interaction of the modified peptides with membrane mimetics. Single or triple 5OHW substitution did not have a large effect on the MIC of the parent peptide against Escherichia coli and Bacillus subtilis. However, the mechanism of action was altered by simultaneously replacing all three Trp with 5OHW. Our results suggest that the inner membrane of Gram-negative bacteria did not constitute the main target of this particular tritrpticin derivative. Since the addition of a hydroxyl group to the indole motif of the Trp residue was able to modify the mechanism of action of the peptides, our data confirm the importance of the Trp cluster in tritrpticin. This work also shows that 5OHW constitutes a new probe to modulate the antimicrobial activity and mechanism of action of other Trp-rich antimicrobial peptides.

Overexpression of Antimicrobial, Anticancer, and Transmembrane Peptides in Escherichia coli through a Calmodulin-Peptide Fusion System.

In recent years, the increasing number of antibiotic-resistant bacteria has become a serious health concern. Antimicrobial peptides (AMPs) are an important component of the innate immune system of most organisms. A better understanding of their structures and mechanisms of action would lead to the design of more potent and safer AMPs as alternatives for current antibiotics. For detailed investigations, effective recombinant production which allows the facile modification of the amino acid sequence, the introduction of unnatural amino acids, and labeling with stable isotopes for nuclear magnetic resonance (NMR) studies is desired. Several expression strategies have been introduced in previous reports; however, their effectiveness has been limited to a select few AMPs. Here, we have studied calmodulin (CaM) as a more universal carrier protein to express many types of AMPs in E. coli. We have discovered that the unique architecture of CaM, consisting of two independent target binding domains with malleable methionine-rich interaction surfaces, can accommodate numerous amino acid sequences containing basic and hydrophobic residues. This effectively masks the toxic antimicrobial activities of many amphipathic AMPs and protects them from degradation during expression and purification. Here, we demonstrate the expression of various AMPs using a CaM-fusion expression system, including melittin, fowlicidin-1, tritrpticin, indolicidin, puroindoline A peptide, magainin II F5W, lactoferrampin B, MIP3α51-70, and human ß-defensin 3 (HBD-3), the latter requiring three disulfide bonds for proper folding. In addition, our approach was extended to the transmembrane domain of the cell adhesion protein l-selectin. We propose the use of the CaM-fusion system as a universal approach to express many cationic amphipathic peptides that are normally toxic and would kill the bacterial host cells.

Native thrombocidin-1 and unfolded thrombocidin-1 exert antimicrobial activity via distinct structural elements.

Chemokines (chemotactic cytokines) can have direct antimicrobial activity, which is apparently related to the presence of a distinct positively charged patch on the surface. However, chemokines can retain antimicrobial activity upon linearization despite the loss of their positive patch, thus questioning the importance of this patch for activity. Thrombocidin-1 (TC-1) is a microbicidal protein isolated from human blood platelets. TC-1 only differs from the chemokine NAP-2/CXCL7 by a two-amino acid C-terminal deletion, but this truncation is crucial for antimicrobial activity. We assessed the structure-activity relationship for antimicrobial activity of TC-1. Reduction of the charge of the TC-1-positive patch by replacing lysine 17 with alanine reduced the activity against bacteria and almost abolished activity against the yeast Candida albicans. Conversely, augmentation of the positive patch by increasing charge density or size resulted in a 2-3-fold increased activity against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis but did not substantially affect activity against C. albicans. Reduction of TC-1 resulted in loss of the folded conformation, but this disruption of the positive patch did not affect antimicrobial activity. Using overlapping 15-mer synthetic peptides, we demonstrate peptides corresponding to the N-terminal part of TC-1 to have similar antimicrobial activity as intact TC-1. Although we demonstrate that the positive patch is essential for activity of folded TC-1, unfolded TC-1 retained antimicrobial activity despite the absence of a positive patch. This activity is probably exerted by a linear peptide stretch in the N-terminal part of the molecule. We conclude that intact TC-1 and unfolded TC-1 exert antimicrobial activity via distinct structural elements.

Investigating the cationic side chains of the antimicrobial peptide tritrpticin: hydrogen bonding properties govern its membrane-disruptive activities.

The positively charged side chains of cationic antimicrobial peptides are generally thought to provide the initial long-range electrostatic attractive forces that guide them towards the negatively charged bacterial membranes. Peptide analogs were designed to examine the role of the four Arg side chains in the cathelicidin peptide tritrpticin (VRRFPWWWPFLRR). The analogs include several noncoded Arg and Lys derivatives that offer small variations in side chain length and methylation state. The peptides were tested for bactericidal and hemolytic activities, and their membrane insertion and permeabilization properties were characterized by leakage assays and fluorescence spectroscopy. A net charge of +5 for most of the analogs maintains their high antimicrobial activity and directs them towards preferential insertion into model bacterial membrane systems with a similar extent of burial of the Trp side chains. However the peptides exhibit significant functional differences. Analogs with methylated cationic side chains cause lower levels of membrane leakage and are associated with lower hemolytic activities, making them potentially attractive pharmaceutical candidates. Analogs containing the Arg guanidinium groups cause more membrane disruption than those containing the Lys amino groups. Peptides in the latter group with shorter side chains have increased membrane activity and conversely, elongating the Arg residue causes slightly higher membrane activity. Altogether, the potential for strong hydrogen bonding between the four positive Arg side chains with the phospholipid head groups seems to be a determinant for the membrane disruptive properties of tritrpticin and many related cationic antimicrobial peptides.

Improved anticancer potency by head-to-tail cyclization of short cationic anticancer peptides containing a lipophilic ß(2,2) -amino acid.

We have recently reported a series of synthetic anticancer heptapeptides (H-KKWß(2,2) WKK-NH(2) ) containing a central achiral and lipophilic ß(2,2) -amino acid that display low toxicity against non-malignant cells and high proteolytic stability. In the present study, we have further investigated the effects of increasing the rigidity and amphipathicity of two of our lead heptapeptides by preparing a series of seven to five residue cyclic peptides containing the two most promising ß(2,2) -amino acid derivatives as part of the central lipophilic core. The peptides were tested for anticancer activity against human Burkitt's lymphoma (Ramos cells), haemolytic activity against human red blood cells (RBC) and cytotoxicity against healthy human lung fibroblast cells (MRC-5). The results demonstrated a considerable increase in anticancer potency following head-to-tail peptide cyclization, especially for the shortest derivatives lacking a tryptophan residue. High-resolution NMR studies and molecular dynamics simulations together with an annexin-V-FITC and propidium iodide fluorescent assay showed that the peptides had a membrane disruptive mode of action and that the more potent peptides penetrated deeper into the lipid bilayer. The need for new anticancer drugs with novel modes of action is demanding, and development of short cyclic anticancer peptides with an overall rigidified and amphipathic structure is a promising approach to new anticancer agents.

Design of a novel tryptophan-rich membrane-active antimicrobial peptide from the membrane-proximal region of the HIV glycoprotein, gp41.

A number of physicochemical characteristics have been described which contribute to the biological activity of antimicrobial peptides. This information was used to design a novel antimicrobial peptide sequence by using an intrinsically inactive membrane-associated peptide derived from the HIV glycoprotein, gp41, as a starting scaffold. This peptide corresponds to the tryptophan-rich membrane-proximal region of gp41, which is known to interact at the interfacial region of the viral membrane and adopts a helical structure in the presence of lipids. Three synthetic peptides were designed to increase the net positive charge and amphipathicity of this 19-residue peptide. Ultimately, the peptide with the greatest degree of amphipathicity and largest positive charge proved to be the most potent antimicrobial, and this peptide could be further modified to improve the antimicrobial activity. However, the other two peptides were relatively ineffective antimicrobials and instead proved to be extremely hemolytic. This work demonstrates a novel approach for the design of unexplored antimicrobial peptide sequences but it also reveals that the biological and cytotoxic activities of these polypeptides depend on a number of interrelated factors.

Seropositivity to SARS-CoV-2 in Alberta, Canada in a post-vaccination period (March 2021-July 2021).

BACKGROUND: The COVID-19 pandemic has necessitated the need to rapidly make public health decisions. We systematically evaluated SARS-CoV-2 seropositivity to understand local COVID-19 epidemiology and support evidence-based public health decision making. METHODS: Residual blood samples were collected for SARS-CoV-2 receptor binding domain (RBD) IgG testing over a 1-5 day period monthly from 26 February 2021-9 July 2021 from six clinical laboratories across the province of Alberta, Canada. Monthly crude and adjusted (for age and gender) seropositivity were calculated. Results were linked to provincial administrative, laboratory, and vaccine databases. RESULTS: 60,632 individual blood samples were tested. Vaccination data were available for 98.8% of samples. Adjusted RBD IgG positivity rose from 11.9% (95% confidence interval [CI] 11.9-12.0%) in March 2021 to 70.2% (95% CI 70.2-70.3%) in July 2021 (p < .0001). Seropositivity rose from 9.4% (95% CI 9.3-9.4%) in March 2021 to 20.2% (95% CI 20.1-20.2%) in July 2021 in unvaccinated Albertans. Unvaccinated seropositive individuals were from geographic areas with significantly (p < .001) lower median household income, lower proportion of married/common-law relationships, larger average household size and higher proportions of visible minorities compared to seronegative unvaccinated individuals. In July 2021, the age groups with the lowest and highest seropositivity in unvaccinated Albertans were those ≥80 years (12.0%, 95% CI 5.3-18.6%) and 20-29 years (24.2%, 95% CI 19.6-28.8%), respectively. Of seropositive unvaccinated individuals, 50.2% (95% CI 45.9-54.5%) had no record of prior SARS-CoV-2 molecular testing. CONCLUSIONS: Longitudinal surveillance of SARS-CoV-2 seropositivity with data linkage is valuable for decision-making during the pandemic.

Structure-function studies of chemokine-derived carboxy-terminal antimicrobial peptides.

Recent reports which show that several chemokines can act as direct microbicidal agents have drawn renewed attention to these chemotactic signalling proteins. Here we present a structure-function analysis of peptides derived from the human chemokines macrophage inflammatory protein-3alpha (MIP-3alpha/CCL20), interleukin-8 (IL-8), neutrophil activating protein-2 (NAP-2) and thrombocidin-1 (TC-1). These peptides encompass the C-terminal alpha-helices of these chemokines, which have been suggested to be important for the direct antimicrobial activities. Far-UV CD spectroscopy showed that the peptides are unstructured in aqueous solution and that a membrane mimetic solvent is required to induce a helical secondary structure. A co-solvent mixture was used to determine solution structures of the peptides by two-dimensional (1)H-NMR spectroscopy. The highly cationic peptide, MIP-3alpha(51-70), had the most pronounced antimicrobial activity and displayed an amphipathic structure. A shorter version of this peptide, MIP-3alpha(59-70), remained antimicrobial but its structure and mechanism of action were unlike that of the former peptide. The NAP-2 and TC-1 proteins differ in their sequences only by the deletion of two C-terminal residues in TC-1, but intact TC-1 is a very potent antimicrobial while NAP-2 is inactive. The corresponding C-terminal peptides, NAP-2(50-70) and TC-1(50-68), had very limited and no bactericidal activity, respectively. This suggests that other regions of TC-1 contribute to its bactericidal activity. Altogether, this work provides a rational structural basis for the biological activities of these peptides and proteins and highlights the importance of experimental characterization of peptide fragments as distinct entities because their activities and structural properties may differ substantially from their parent proteins.

Exploring platelet chemokine antimicrobial activity: nuclear magnetic resonance backbone dynamics of NAP-2 and TC-1.

The platelet chemokines neutrophil-activating peptide-2 (NAP-2) and thrombocidin-1 (TC-1) differ by only two amino acids at their carboxy-terminal ends. Nevertheless, they display a significant difference in their direct antimicrobial activities, with the longer NAP-2 being inactive and TC-1 being active. In an attempt to rationalize this difference in activity, we studied the structure and the dynamics of both proteins by nuclear magnetic resonance (NMR) spectroscopy. Using 15N isotope-labeled protein, we confirmed that the two monomeric proteins essentially have the same overall structure in aqueous solution. However, NMR relaxation measurements provided evidence that the negatively charged carboxy-terminal residues of NAP-2 experience a restricted motion, whereas the carboxy-terminal end of TC-1 moves in an unrestricted manner. The same behavior was also seen in molecular dynamic simulations of both proteins. Detailed analysis of the protein motions through model-free analysis, as well as a determination of their overall correlation times, provided evidence for the existence of a monomer-dimer equilibrium in solution, which seemed to be more prevalent for TC-1. This finding was supported by diffusion NMR experiments. Dimerization generates a larger cationic surface area that would increase the antimicrobial activities of these chemokines. Moreover, these data also show that the negatively charged carboxy-terminal end of NAP-2 (which is absent in TC-1) folds back over part of the positively charged helical region of the protein and, in doing so, interferes with the direct antimicrobial activity.

Factors Associated with Hyponatremia in Patients Newly Prescribed Citalopram: A Retrospective Observational Study.

BACKGROUND: Hyponatremia is a common and under-recognized adverse drug reaction of selective serotonin re-uptake inhibitor (SSRI) antidepressants. Despite its clinical importance, there are few large-scale studies on the factors associated with hyponatremia. OBJECTIVE: The aim of this study was to determine the incidence of hyponatremia and to identify patient factors associated with hyponatremia in a large, population-based cohort initiating new prescriptions for citalopram. METHODS: We included all patients with a new prescription for citalopram during 2010-2017, inclusive, with baseline and post-initiation serum sodium values available. Data were obtained from an Alberta Health Pharmacy database to identify new citalopram prescriptions. Laboratory values for patients with new prescriptions were obtained from linked Calgary Laboratory Services data. Incident hyponatremia was defined as serum sodium level < 135 mmol/L, following prescription initiation. Associations were determined by performing Cox regression with time-varying covariate analysis, with the development of hyponatremia as the dependent variable. RESULTS: A total of 19,679 patients with new prescriptions were identified; 12,842 females and 6837 males. The mean age was 55.48 years (SD 21.35). Of these patients, 3250 (16.5%) developed hyponatremia, 1996 (15.5% of) females and 1254 (18.3% of) males (p = 0.002). Cox regression showed significant associations of hyponatremia with lower baseline sodium (HR 0.788), older age (HR 1.029), thiazide diuretic use (HR 1.141), and male sex (HR 1.168). Pharmaceutical manufacturer or strength of citalopram did not have significant effects on the development of hyponatremia. CONCLUSION: This study provides additional data on the predictors of hyponatremia among patients initiating citalopram therapy. We report a 16.5% incidence of hyponatremia after starting citalopram treatment, and significant new findings include a higher incidence in males. This is the first published incidence of hyponatremia following the initiation of citalopram treatment across all ages in Canada.

Pre-Vaccine Positivity of SARS-CoV-2 Antibodies in Alberta, Canada during the First Two Waves of the COVID-19 Pandemic.

We systematically evaluated SARS-CoV-2 IgG positivity in a provincial cohort to understand the local epidemiology of COVID-19 and support evidence-based public health decisions. Residual blood samples were collected for serology testing over 5-day periods monthly from June 2020 to January 2021 from six clinical laboratories across the province of Alberta, Canada. A total of 93,993 individual patient samples were tested with a SARS-CoV-2 nucleocapsid antibody assay with positives confirmed using a spike antibody assay. Population-adjusted SARS-CoV-2 IgG seropositivity was 0.92% (95% confidence interval [CI]: 0.91 to 0.93%) shortly after the first COVID-19 wave in June 2020, increasing to 4.63% (95% CI: 4.61 to 4.65%) amid the second wave in January 2021. There was no significant difference in seropositivity between males and females (1.39% versus 1.27%; P = 0.11). Ages with highest seropositivity were 0 to 9 years (2.71%, 95% CI: 1.64 to 3.78%) followed by 20 to 29 years (1.58%, 95% CI: 1.12 to 2.04%), with the lowest rates seen in those aged 70 to 79 (0.79%, 95% CI: 0.65 to 0.93%) and >80 (0.78%, 95% CI: 0.60 to 0.97%). Compared to the seronegative group, seropositive patients inhabited geographic areas with lower household income ($87,500 versus $97,500; P < 0.001), larger household sizes, and higher proportions of people with education levels of secondary school or lower, as well as immigrants and visible minority groups (all P < 0.05). A total of 53.7% of seropositive individuals were potentially undetected cases with no prior positive COVID-19 nucleic acid test (NAAT). Antibodies were detectable in some patients up to 9 months post positive NAAT result. This seroprevalence study will continue to inform public health decisions by identifying at-risk demographics and geographical areas. IMPORTANCE Using SARS-CoV-2 serology testing, we assessed the proportion of people in Alberta, Canada (population 4.4 million) positive for COVID-19 antibodies, indicating previous infection, during the first two waves of the COVID-19 pandemic (prior to vaccination programs). Linking these results with sociodemographic population data provides valuable information as to which groups of the population are more likely to have been infected with the SARS-CoV-2 virus to help facilitate public health decision-making and interventions. We also compared seropositivity data with previous COVID-19 molecular testing results. Absence of antibody and molecular testing were highly correlated (95% negative concordance). Positive antibody correlation with a previous positive molecular test was low, suggesting the possibility of mild/asymptomatic infection or other reasons leading individuals from seeking medical attention. Our data highlight that the true estimate of population prevalence of COVID-19 is likely best informed by combining data from both serology and molecular testing.

Title Assessing Potentially Inappropriate Medications in Seniors: Differences between American Geriatrics Society and STOPP Criteria, and Preventing Adverse Drug Reactions.

Key problems for seniors are their exposure to "potentially inappropriate medications" and "potential medication omissions", which place them at risk for moderate, severe, or fatal adverse drug reactions. This study of 82,935 first admissions to acute care hospitals in Calgary during 2013-2018 identified 294,160 Screening Tool of Older People's Prescriptions (STOPP) potentially inappropriate medications (PIMs) (3.55/patient), 226,970 American Geriatric Society (AGS) Beers PIMs (2.74/patient), 59,396 START potential prescribing omissions (PPOs) (0.72/patient), and 85,288 STOPP PPOs (1.03/patient) for which a new prescription corrected the omission. This represents an overwhelming workload to prevent inappropriate prescriptions continuing during the hospitalisation and then deprescribe them judiciously. Limiting scrutiny to the most frequent PIMs and PPOs will identify many moderate, severe, or fatal risks of causing adverse drug reactions (ADRs) but to identify all PIMs or PPO involving moderate or severe risks of ADRs also involves searching lower in the frequency list of patients. Deciding whether to use the STOPP or AGS Beers PIM lists is an important issue in searching for ADRs, because the Pearson correlation coefficient for agreement between the STOPP and AGS Beers PIM totals in this study was 0.7051 (95% CI 0.7016 to 0.7085; p < 0.001). The combined lists include 289 individual PIM medications but STOPP and AGS have only 159 (55%) in common. The AGS Beers lists include medications used in the US and STOPP/START those used in Europe. The AGS authors recommend using both criteria. The ideal solution to the problem is to implement carefully constructed Clinical Decision Support Systems (CDSS) as in the SENATOR trial, then for an experienced pharmacist to focus on the key PIMs and PPOs likely to lead to moderate, severe, or fatal ADRs. The pharmacist and key decision makers on the services need to establish a collegial relationship to discuss frequently changing the medications that place the patients at risk. Then, the remaining PIMs and PPOs that relate to chronic disease management can be discussed by phone with the family physician using the discharge summary, which lists the medications for potential deprescribing.

Potentially Inappropriate Prescribing and Potential Prescribing Omissions in 82,935 Older Hospitalised Adults: Association with Hospital Readmission and Mortality Within Six Months.

Polypharmacy with "potentially inappropriate medications" (PIMs) and "potential prescribing omissions" (PPOs) are frequent among those 65 and older. We assessed PIMs and PPOs in a retrospective study of 82,935 patients ≥ 65 during their first admission in the period March 2013 through February 2018 to the four acute-care Calgary hospitals. We used the American Geriatric Society (AGS) and STOPP/START criteria to assess PIMs and PPOs. We computed odds ratios (ORs) for key outcomes of concern to patients, their families, and physicians, namely readmission and/or mortality within six months of discharge, and controlled for age, sex, numbers of medications, PIMs, and PPOs. For readmission, the adjusted OR for number of medications was 1.09 (1.09-1.09), for AGS PIMs 1.14 (1.13-1.14), for STOPP PIMs 1.15 (1.14-1.15), for START PPOs 1.04 (1.02-1.06), and for START PPOs correctly prescribed 1.16 (1.14-1.17). For mortality within 6 months of discharge, the adjusted OR for the number of medications was 1.02 (1.01-1.02), for STOPP PIMs 1.07 (1.06-1.08), for AGS PIMs 1.11 (1.10-1.12), for START PPOs 1.31 (1.27-1.34), and for START PPOs correctly prescribed 0.97 (0.94-0.99). Algorithm rule mining identified an 8.772 higher likelihood of mortality with the combination of STOPP medications of duplicate drugs from the same class, neuroleptics, and strong opioids compared to a random relationship, and a 2.358 higher likelihood of readmission for this same set of medications. Detailed discussions between patients, physicians, and pharmacists are needed to improve these outcomes.

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes.

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from +4 to +5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an alpha-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.

Expression and Purification of Chemokine MIP-3α (CCL20) through a Calmodulin-Fusion Protein System.

Human macrophage inflammatory protein 3α (MIP-3α), also known as CCL20, is a 70 amino acid chemokine that selectively binds and activates chemokine receptor 6 (CCR6). This chemokine is responsible for inducing the migration of immature dendritic cells, effector, or memory T-cells, and B-cells. Moreover, the MIP-3α protein has been shown to display direct antimicrobial, antiviral and antiprotozoal activities. Because of the potential therapeutic uses of this protein, the efficient production of MIP-3α is of great interest. However, bacterial recombinant production of the MIP-3α protein has been limited by the toxicity of this extremely basic protein (pI 9.7) toward prokaryotic cells, and by solubility problems during expression and purification. In an attempt to overcome these issues, we have investigated the bacterial recombinant expression of MIP-3α by using several common expression and fusion tags, including 6× histidine (His), small ubiquitin modifier protein (SUMO), thioredoxin (TRX), ketosteroid isomerase (KSI), and maltose binding protein (MBP). We have also evaluated a recently introduced calmodulin (CaM)-tag that has been used for the effective expression of many basic antimicrobial peptides (AMPs). Here, we show that the CaM fusion tag system effectively expressed soluble MIP-3α in the cytoplasm of Escherichia coli with good yields. Rapid purification was facilitated by the His-tag that was integrated in the CaM-fusion protein system. Multidimensional nuclear magnetic resonance (NMR) studies demonstrated that the recombinant protein was properly folded, with the correct formation of disulfide bonds. In addition, the recombinant MIP-3α had antibacterial activity, and was shown to inhibit the formation of Pseudomonas aeruginosa biofilms.

Dataset of clinical laboratory tests according to ordering variance among family physicians in Calgary, Alberta, Canada.

This data incorporates 2016 testing volumes ordered by family physicians and performed at Calgary Laboratory Services (CLS), the sole supplier of clinical laboratory services for the catchment area of the City of Calgary, Alberta, Canada. For each test, the mean number of tests ordered per patient was calculated over ordering Calgary physicians, along with arithmetic coefficients of variation (CV's). The latter parameter is reflective of variance in ordering practice among family physicians practicing in Calgary and is proposed as a benchmark measure for laboratory utilization in our accompanying research article [1]. The data table encompasses 358 tests ordered by at least 3 family physicians at a minimum total frequency of 100 within the 2016 study period and is presented in ascending order of rank in CV.

Evaluating practice variance among family physicians to identify targets for laboratory utilization management.

BACKGROUND: There is widespread variation in testing practice among practitioners, however there has been no objective way to pinpoint target tests for utilization management. We propose to take advantage of inter-physician variance in clinical practice as a quantitative measure to generate lists of potentially misutilized tests. METHODS: Testing frequencies from a database of clinical testing volumes for outpatients in Calgary, Canada, were obtained for the study period of 2016. For each chemistry, microbiology or hematology test, an arithmetic coefficient of variation (CV) was calculated from family physicians' ordering frequencies. RESULTS: The mean CV for all 358 tests considered was 219% (95% CI 206-231%) with a range of 52-729%. The highest variance was observed for human T-lymphotropic virus antibody testing and several tests for heavy metal levels (mercury, copper, zinc and chromium). Among the 100 most commonly run tests, high variance was found for several endocrinology tests including cortisol. CONCLUSIONS: The utility of ranking clinical tests by ordering variance presents a practical approach to evaluate relative variation in physician practice strategy and to identify potential areas of misutilization.

Incidence of acute lymphocytic leukemia in Calgary, Alberta, Canada: a retrospective cohort study.

OBJECTIVE: Acute lymphocytic leukemia (ALL) is a rare malignant neoplasm that develops from abnormal lymphoid stem cells. ALL incidence is highest among children and declines towards adolescence. There is limited data on the epidemiology of ALL, especially in Canada. This retrospective cohort study used patient data from the Calgary Laboratory Services Cancer Cytogenetics Laboratory to report the incidence rate of ALL in Calgary, Alberta, Canada. New cases of ALL were identified for the 5-year period of January 1, 2011 until December 31, 2015. Reported incidence rates were categorized by sex and age groups, and age-standardized to the Canadian population. RESULTS: There were an average of 11.4 new cases of ALL diagnosed per year between 2011 and 2015. The total incidence rate per 100,000 person-years was 0.84. Incidence rates peaked in children aged 0-4 with 7.55 and 3.32 cases per 100,000 person-years for males and females, respectively. The median age of diagnosis was 8 years. Incidence rates were generally lowest for adults aged 20 and over. The ratio of males to females diagnosed with ALL was 1.59. Overall, the recent incidence of ALL in Calgary is comparatively low with a preference for males and children below 5 years of age.

Structural and dynamic characterization of a freestanding acyl carrier protein involved in the biosynthesis of cyclic lipopeptide antibiotics.

Friulimicin is a cyclic lipodecapeptide antibiotic that is produced by Actinoplanes friuliensis. Similar to the related lipopeptide drug daptomycin, the peptide skeleton of friulimicin is synthesized by a large multienzyme nonribosomal peptide synthetase (NRPS) system. The LipD protein plays a major role in the acylation reaction of friulimicin. The attachment of the fatty acid group promotes its antibiotic activity. Phylogenetic analysis reveals that LipD is most closely related to other freestanding acyl carrier proteins (ACPs), for which the genes are located near to NRPS gene clusters. Here, we report that the solution NMR structure of apo-LipD is very similar to other four-helix bundle forming ACPs from fatty acid synthase (FAS), polyketide synthase, and NRPS systems. By recording NMR dynamics data, we found that the backbone motions in holo-LipD are more restricted than in apo-LipD due to the attachment of phosphopantetheine moiety. This enhanced stability of holo-LipD was also observed in differential scanning calorimetry experiments. Furthermore, we demonstrate that, unlike several other ACPs, the folding of LipD does not depend on the presence of divalent cations, although the presence of Mg2+ or Ca2+ can increase the protein stability. We propose that small structural rearrangements in the tertiary structure of holo-LipD which lead to the enhanced stability are important for the cognate enzyme recognition for the acylation reaction. Our results also highlight the different surface charges of LipD and FAS-ACP from A. friuliensis that would allow the acyl-CoA ligase to interact preferentially with the LipD instead of binding to the FAS-ACP.

Influence of diurnal variation and fasting on serum iron concentrations in a community-based population.

OBJECTIVES: Serum iron is an important clinical test to help identify cases of iron deficiency or overload. Fluctuations caused by diurnal variation and diet are thought to influence test results, which may affect clinical patient management. We examined the impact of these preanalytical factors on iron concentrations in a large community-based cohort. DESIGN AND METHODS: Serum iron concentration, blood collection time, fasting duration, patient age and sex were obtained for community-based clinical testing from the Laboratory Information Service at Calgary Laboratory Services for the period of January 2011 to December 2015. RESULTS: A total of 276,307 individual test results were obtained. Iron levels were relatively high over a long period from 8:00 to 15:00. Mean concentrations were highest at blood collection times of 11:00 for adult men and 12:00 for adult women and children, however iron levels peaked as late as 15:00 in teenagers. With regard to fasting, iron levels required approximately 5h post-prandial time to return to a baseline, except for children and teenage females where no significant variation was seen until after 11h fasting. After 10h fasting, iron concentrations in all patient groups gradually increased to higher levels compared to earlier fasting times. CONCLUSIONS: Serum iron concentrations remain reasonably stable during most daytime hours for testing purposes. In adults, blood collection after 5 to 9h fasting provides a representative estimate of a patient's iron levels. For patients who have fasted overnight, i.e. ≥12h fasting, clinicians should be aware that iron concentrations may be elevated beyond otherwise usual levels.